Systemic gene delivery of p53 with cationic liposomes has been shown to reduce tumor growth in pre-clinical models. Although gene therapy with p53 may include several antiturnor mechanisms, antiangiogenesis is one important mechanism by which p53 inhibits tumor growth. Similar to other genes encoding antiangiogenic proteins, p53 gene therapy delivered by cationic liposomes is hampered by inadequate transfection in vivo. Recently our laboratory has developed linear and branched HK (histidine-lysine) polymers that in combination with liposomes significantly increased transfection in the presence or absence of serum. To test the overall hypothesis that further understanding of HK enhancement of transfection will facilitate the development of HK carriers for in vivo antiangiogenic gene therapy, the following specific aims are planned. Aim 1 will compare liposomes to HK/liposomes as gene delivery carriers of p53 for their ability to inhibit tumor growth. Since the tumor vasculature is readily accessible to systemic therapy, targeting tumor endothelial cells increases the likelihood that gene therapy will be successful. Two promising HK/liposome/p53 plasmid complexes will be compared to a liposome/p53 plasmid complex for their ability to reduce tumor angiogenesis and growth in vivo. To enhance specificity and antitumor efficacy of p53 in vivo, these HK-containing carriers will be modified with a vascular specific cyclic peptide (-NGR-) and a hydrophilic shield (PEG). Because the tumor endothelium is a primary target of complexes in vivo, we will test our hypothesis that the addition of HK polymers to liposome/p53 plasmid complexes will significantly improve gene therapy resulting in greater tumor inhibition. Since instability of the complexes in serum limits in vivo efficacy, Aim 2 is designed to clarify the mechanism by which the HK polymer enhances stability of liposome:DNA complexes in the presence of serum. In contrast to poly-L-lysine, the presence of HK maintains high level of gene expression and markedly enhances resistance of the liposome:DNA complex to serum, even with prolonged incubations. Whether hydrogen bonding or hydrophobic properties of histidine stabilize the HK:liposome:DNA complexes in the presence of serum will be tested. In Aim 3 the role of binding of HK to DNA in transfection will be examined to test the hypothesis that the interplay between different affinities of these polymers with DNA and the cell's endosomal pH accounts for variations in transfection efficiency. The ability to alter the histidine and lysine ratio and the complexity of the HK polymer is likely to change endosomal pH, which may affect binding and transfection efficiency. Understanding these properties that govern DNA release from the HK/liposome carrier within acidic endosomes will enable the development of improved carriers. Aim 4 is designed to use information gathered in the above aims to compare the derivatives of HK for their abilities to inhibit tumor growth. Based on the mechanistic studies determined in the previous aims, improved designs of the HK carrier will be developed. In combination with modified liposomes, several HK polymers will be compared with the optimal carrier in Aim 1 for their ability to augment the antitumor activity of p53 in vivo. The modified HK-containing carriers of p53 will be investigated for their antitumor activity of lung metastases and orthotopically implanted tumors.